ORIGINAL RESEARCH ARTICLE published: 13 January 2014 doi: 10.3389/fonc.2013.00333

Evidence that GRIN2A mutations in melanoma correlate with decreased survival Stacey Ann N. D’mello 1 , Jack U. Flanagan 2,3 , Taryn N. Green 1 , Euphemia Y. Leung 2 , Marjan E. Askarian-Amiri 2 , Wayne R. Joseph 2 , Michael R. McCrystal 4,5 , Richard J. Isaacs 6 , James H. F. Shaw 7 , Christopher E. Furneaux 8 , Matthew J. During 9,10 , Graeme J. Finlay 2 , Bruce C. Baguley 2 and Maggie L. Kalev-Zylinska 1,11 * 1

Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand Auckland Cancer Society Research Centre, University of Auckland, Auckland, New Zealand 3 Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand 4 Department of Clinical Oncology, Auckland District Health Board, Auckland, New Zealand 5 Canopy Cancer Care, Mercy Hospital, Auckland, New Zealand 6 Regional Cancer Treatment Service, Palmerston North Public Hospital, Palmerston North, New Zealand 7 Oncology Surgery Ltd., Auckland, New Zealand 8 Department of Neurosurgery, Auckland District Health Board, Auckland, New Zealand 9 Department of Molecular Virology, Immunology and Medical Genetics, Neuroscience and Neurological Surgery, Ohio State University, Columbus, OH, USA 10 Centre for Brain Research, University of Auckland, Auckland, New Zealand 11 LabPlus Haematology, Auckland District Health Board, Auckland, New Zealand 2

Edited by: Sven Bilke, National Institutes of Health, USA Reviewed by: Josh Waterfall, National Institutes of Health, USA Anna DeFazio, University of Sydney at Westmead Millennium Institute, Australia *Correspondence: Maggie L. Kalev-Zylinska, Department of Molecular Medicine and Pathology, University of Auckland, 85 Park Road, Grafton, Auckland, ACM 1142, New Zealand e-mail: [email protected]

Previous whole-exome sequencing has demonstrated that melanoma tumors harbor mutations in the GRIN2A gene. GRIN2A encodes the regulatory GluN2A subunit of the glutamate-gated N-methyl-D-aspartate receptor (NMDAR), involvement of which in melanoma remains undefined. Here, we sequenced coding exons of GRIN2A in 19 lowpassage melanoma cell lines derived from patients with metastatic melanoma. Potential mutation impact was evaluated in silico, including within the GluN2A crystal structure, and clinical correlations were sought. We found that of 19 metastatic melanoma tumors, four (21%) carried five missense mutations in the evolutionarily conserved domains of GRIN2A; two were previously reported. Melanoma cells that carried these mutations were treatment-naïve. Sorting intolerant from tolerant analysis predicted that S349F, G762E, and P1132L would disrupt protein function. When modeled into the crystal structure of GluN2A, G762E was seen to potentially alter GluN1–GluN2A interactions and ligand binding, implying disruption to NMDAR functionality. Patients whose tumors carried non-synonymous GRIN2A mutations had faster disease progression and shorter overall survival (P < 0.05). This was in contrast to the BRAF V600E mutation, found in 58% of tumors but showing no correlation with clinical outcome (P = 0.963). Although numbers of patients in this study are small, and firm conclusions about the association between GRIN2A mutations and poor clinical outcome cannot be drawn, our results highlight the high prevalence of GRIN2A mutations in metastatic melanoma and suggest for the first time that mutated NMDARs impact melanoma progression. Keywords: melanoma, GRIN2A, mutation, GluN2A, NMDAR, NMDA receptor, glutamate, prognosis

INTRODUCTION The genomic revolution of recent years has led to substantial advances in the cataloging of mutations in melanoma, most notable of which have been activating mutations in BRAF, NRAS, and KIT genes (1). The presence of these mutations helps to guide treatment with RAF, MEK, and KIT inhibitors, but they do not predict disease progression or survival of patients (2). In general, the usefulness of molecular biomarkers in determining prognosis in melanoma remains limited. Mutations in GRIN2A have been reported in up to a third of melanoma samples (3), although there is wide variation between studies (4–7) and no data on their clinical relevance. The GRIN2A gene, located on chromosome 16p13.2, encodes the GluN2A

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protein, a regulatory subunit of the glutamate-gated N -methyl-daspartate receptor (NMDAR) (8, 9). NMDARs are best known for their roles in the brain; hence, the finding of GRIN2A mutations in melanoma had been unexpected. Nevertheless, NMDARs have attracted attention for their potential contribution in cancer due to the effects on cell death, survival, and migration (10, 11). Excessive NMDAR activation overloads the cell with calcium and leads to cell death (12). On the other hand, normal NMDAR activity promotes cell survival through the phosphatidylinositol 3-kinase (PI3-K) and extracellular signal-regulated kinase (ERK) signaling pathways (13). In addition, NMDAR effects on cell migration may affect tumor spread in tissue (10). Current knowledge on the NMDARs in the context of melanoma is limited, although expression of

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GluN2A in both normal and malignant melanocytes has been demonstrated (14, 15). NMDAR inhibitors reduce migration and proliferation of melanoma cells in vitro (15). In response to the previously published exome sequencing data (3), we have investigated the prevalence of GRIN2A mutations in 19 low-passage metastatic melanoma cell lines out of over a 100 developed in our institution, and retrospectively correlated the presence of GRIN2A mutations with patient outcome.

MATERIALS AND METHODS PATIENTS AND TUMOR MATERIAL

Low-passage melanoma cell lines derived from 19 patients with metastatic melanoma treated at two independent national sites were used in this study (Table S1 in Supplementary Material). Written informed consent was obtained from all participants prior to enrolment; all study procedures were approved by Northern A Health and Disability Human Ethics Committee. This was not a clinical trial, and study procedures did not affect patient management in any way. Patients underwent skin, lymph node, or distant organ biopsies for diagnosis, staging, or treatment, as required clinically. Excess tissue was used to establish melanoma cell lines, as described (16). Cell lines for sequencing were chosen randomly out of over a 100 previously established in our center; all were passaged T

L794L

13

Homo-

C >T

F1344F

14

Hetero-

A1409A

14

Hetero-

g.419243

g.418606 C >T

4

c.4032

c.3395

003

F186F

g.413689

g.417877 C >T

C >T

c.2380

c.2666

100

Hetero-

g.244347

g.384407 G>A

4

c.558

c.2285

007

F177F

g.244320

g.291693 G>A

Zygosity

c.531

c.1046

061

Exon

086 P1133S

14

Hetero-

0

3.32

A>C c.4227

c.3397

g.419438

g.418608 Mutations are listed in the order of location along the sequence. Mutations are listed in the order of location along the sequence. Deleterious

c, cDNA; g, genomic DNA; hetero-, heterozygous; homo-, homozygous.

substitutions were predicted from SIFT scores ≤0.05. c, cDNA; g, genomic DNA; hetero-, heterozygous; SIFT, sorting intolerant from tolerant analysis.

the GluN2A protein: C-terminal, N-terminal, and the S2 segment, respectively (Figure 1). The S2 segment forms the ligand binding domain, and the intracytoplasmic C-terminus is involved in intracellular signaling and interactions with the cytoskeleton. Two mutations, G889E and P1132L, were previously reported [Ref. (3, 17), respectively]. SIFT analysis predicted that S349F, G762E, and P1133S would deleteriously affect protein function (Table 3). Five synonymous mutations were also detected (Table 4), as well as four SNPs (Table 5); SNPs were excluded from further analysis. F186F synonymous mutation was found in the NZM 040 cell line derived from patient who received two cycles of POC chemotherapy 8 months prior to cell line derivation (Table S1 in Supplementary Material). In this instance, the possibility that therapy contributed to the presence of this mutation could not be ruled out. We modeled the G762E missense substitution into the 3dimensional X-ray crystal structure of the GluN1–GluN2A heterodimer, 2A5T (22) (Figure 2). This revealed that G762E was located in the distal “hinge” of the glutamate-binding clam-shelllike region of GluN2A. In this location, the mutated glutamate residue was seen to interact with K531 in the GluN1 protein interfacing GluN2A in this region. While K531 formed a hydrogen bond with the backbone carbonyl of F524 in GluN2A, its proximity to the mutated glutamate side-chain (G762E) indicated the potential for new electrostatic interactions between G762E (in GluN2A) and K531 (in GluN1) (Figure 2) with the ability to alter interactions between GluN2A and GluN1 subunits and consequently, impact NMDAR functionality. Conformational changes

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Table 5 | GRIN2A SNPs in tumor-derived cell lines. NZM cell

Substitution and

Amino

line

nucleotide number

acid

Exon

Zygosity

011

G>A

007

c.1275

L425L

6

Homo-

055

g.332946

001

G>A

034

c.1275

L425L

6

Hetero-

061

g.332946

011

G>C

007

c.2085

R695R

11

Homo-

055

g.360408

01

G>C

061

c.2085

R695R

11

Hetero-

W730W

12

Homo-

N1076K

14

Hetero-

g.360408 006

C >T c.2190 g.384312

003

C>A c.3228 g.418439

c, cDNA; g, genomic DNA; homo-, homozygous; hetero-, heterozygous.

that developed to accommodate G762E could also affect ligand binding, as the residue preceding G762E (Y761) was part of the glutamate-binding site (Figure 2) (26).

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CLINICAL ASSOCIATIONS OF GRIN2A MUTATIONS

FIGURE 2 | Model of G762E within the GluN1–GluN2A X-ray crystal structure. The S1S2 loop of GluN2A (in green) is shown interfacing with GluN1 (in cyan). A portion of the GluN2A agonist-binding site is on the left together with bound glutamate (Glut). G762E and F524 residues of GluN2A are 2.7 and 3.9 Å away from K531 in GluN1, respectively; potential new electrostatic interactions are indicated as dashed lines. Nitrogen atoms are in blue, and oxygen in red.

There was no difference in age and gender between patients whose melanoma lines carried GRIN2A mutations and those who did not (Table 2). Two patients in this study presented with disseminated melanoma and both were found to carry nonsynonymous mutations in GRIN2A. The other two patients with the non-synonymous GRIN2A mutations presented with skin lesions that spread to lymph nodes within 9 and 27 months, compared with a median of 37 (0–140) months for patients with non-mutated GRIN2A (P = 0.041; Table 2). Overall, patients with non-synonymous GRIN2A mutations had faster progression of melanoma from skin lesions to the involvement of lymph nodes (P = 0.04) or distant organs (P = 0.012), and shorter overall survival (P = 0.013) compared with patients with non-mutated GRIN2A (Table 2; Figures 3 and 4A). The BRAF V600E mutation was found in 11 of 19 (58%) tumor samples but in contrast to GRIN2A, its presence showed no correlation with overall survival (P = 0.963; Figure 4B; Table S1 in Supplementary Material). Seven patients in this study received systemic therapy – five had chemotherapy and another two autologous tumor vaccine (Table 2). These were in addition to surgery and involved field radiotherapy used in all patients. When compared with patients managed with surgery and radiotherapy alone, the administration of systemic therapy did not change disease spread to lymph nodes (P = 0.849), distant organs (P = 0.499), or overall survival (P = 0.843). Patients with isolated synonymous GRIN2A mutations (n = 3) displayed a trend for poorer outcome, compared with patients with non-mutated GRIN2A, but this was not statistically significant (Figures 3 and 4).

FIGURE 3 | Times of disease progression from diagnosis to lymph node (A) or distant organ (B) metastases for individual patients according to the presence or absence of GRIN2A mutations. Levels of statistical significance are shown. *Progression data for one patient with non-mutated GRIN2A was not available.

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FIGURE 4 | Overall survival according to the GRIN2A (A) or BRAF V600E (B) mutation status. Levels of statistical significance are shown. *Data for one patient with non-mutated GRIN2A (V600E absent) was not available.

DISCUSSION This study has shown that cell lines derived from 4 of 19 (21%) patients with metastatic melanoma carried five missense mutations in GRIN2A. They occurred in three of the four evolutionarily conserved domains of the GluN2A subunit of the NMDAR: the N-terminal, glutamate-binding, and C-terminal domains. All missense mutations were found in treatment-naïve samples. The S349F, G762E, and P1132L substitutions were predicted by SIFT to disrupt protein function. When modeled into the crystal structure of GluN2A, the G762E substitution was seen to potentially alter GluN1–GluN2A interactions and ligand binding, implying disruption to the NMDAR functionality. Patients whose tumors carried non-synonymous mutations in GRIN2A had faster disease progression and shorter overall survival. Our findings suggest for the first time, to our knowledge, that GRIN2A mutations may drive melanoma progression. Our results are in agreement with the seminal whole-exome sequencing work, where non-synonymous mutations in GRIN2A

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GRIN2A mutations in melanoma

were found in 26% of melanoma samples (3). Other exome-wide sequencing projects detected GRIN2A mutations at lower frequencies (4–7). The true frequency of GRIN2A mutations in melanoma remains uncertain, but our results support the high prevalence of GRIN2A mutations in metastatic melanoma (3). Possible causes for differences between studies include the stage of tumors tested, or biological heterogeneity of tumors influenced by demographic, geographical, or environmental factors. Our study demonstrates for the first time that patients with GRIN2A mutations may have more aggressive disease. Considering that there is currently no reliable genetic biomarker that predicts melanoma progression, GRIN2A mutation testing may offer valuable prognostic information. Earlier detection of highly aggressive tumors could assist faster introduction of new therapies for melanoma patients. We propose that the GRIN2A mutation testing be incorporated in larger prospective studies for further evaluation of our findings. Our work has obvious limitations. Patient numbers are small, and the clinical outcome was assessed retrospectively. We did not have access to non-diseased patient DNA to exclude germline polymorphisms (SNPs were excluded using online databases). Original tumor tissue is no longer available to confirm that these contained GRIN2A mutations found in cell lines, but an error in cell line authentication is extremely unlikely. Short tandem repeat profiling has been conducted on NZM cell lines to ensure authentication is possible in the future; however, profiles of original tumors are not available. Small sample size limits our conclusions, and confounders cannot be excluded. Nevertheless, the BRAF mutation status and systemic therapy had no effect on clinical outcome in these patients. Other possible limitations include the relatively late stage of patients at presentation and the selection of cell lines tested. Our success rate of establishing melanoma cell lines is at least 90%, but the possibility of bias toward melanomas that can be grown in culture cannot be ruled out. Synonymous mutations in GRIN2A associated with poorer patient outcome; however, these observations were not statistically significant. Recent studies indicate that synonymous mutations may be important in cancer, primarily through mechanisms that affect RNA processing and protein translation (27, 28). Further work in this area will be required to clarify if synonymous GRIN2A mutations play a role in melanoma biology. Our results have strong implications for basic research. The roles of NMDAR-mediated pathways in melanoma are still unknown and will require elucidation. Well-characterized melanoma cell lines with known mutations, such as those described in this manuscript, will be valuable tools to examine the mechanisms of action and consequences of specific GRIN2A mutations in melanoma tumors. We hypothesize that possible mechanisms through which G762E and other GRIN2A mutations interfere with the NMDAR include reduced NMDAR channel function and disturbed intracellular signaling downstream. Such effects would be most relevant under conditions of NMDAR overactivation, where excessive calcium uptake induces cell toxicity. The lack of NMDAR-mediated cell death could facilitate tumor progression. Our hypothesis is consistent with the previously suggested role for the NMDAR as a tumor suppressor (29). Other GluN subunits (if expressed in melanoma cells) could compensate for the GluN2A disruption or contribute additional functionality.

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NMDAR-mediated pro-cell-survival signaling could also provide oncogenic effects, in keeping with the functional dichotomy of the NMDAR (11). In conclusion, our study suggests that non-synonymous mutations in GRIN2A are present in approximately 20% of patients with metastatic melanoma and associate with faster disease progression and shorter overall survival. The most direct clinical implication of our work is that GRIN2A mutation status may allow earlier detection and hence faster treatments of patients with aggressive tumors. Our data also imply that NMDAR may be a novel molecular modifier in melanoma; hence, further studies into its biological role should be pursued.

AUTHOR CONTRIBUTIONS Stacey Ann N. D’mello conducted experimental work, analyzed data, and wrote the manuscript together with Maggie L. KalevZylinska; Jack U. Flanagan supervised structural modeling of mutation impact; Taryn N. Green, Euphemia Y. Leung, Marjan E. Askarian-Amiri, and Wayne R. Joseph provided advice and assisted experimental procedures; Michael R. McCrystal provided clinical advice; Richard J. Isaacs, James H. F. Shaw, and Christopher E. Furneaux contributed patient samples; Matthew J. During provided mentorship and advice; Graeme J. Finlay provided supervision and edited the manuscript; Bruce C. Baguley helped to design the study, provided supervision, and advice; Maggie L. Kalev-Zylinska designed the study, obtained and analyzed clinical data, provided supervision, and wrote the manuscript together with Stacey Ann N. D’mello. ACKNOWLEDGMENTS Sequencing was performed by Genetic Analysis Services at the University of Otago. Dr. Donald R. Love facilitated additional BRAF mutation testing at LabPlus, Auckland District Health Board. Alexander Trevarton advised on bioinformatics approaches and Nicholas Knowlton on statistical analysis. Funding: This study was supported by Auckland Medical Research Foundation (UOA3700909); Cancer Society (UOA3702269) and Glenn Family Foundation. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at http://www.frontiersin.org/Journal/10.3389/fonc.2013. 00333/abstract Figure S1 | Summary of disease progression events for patients with no, synonymous, and non-synonymous mutations in GRIN2A. Data points for individual patients are shown; horizontal lines mark median values in each group. Levels of statistical difference between groups are shown. *Disease progression data for one patient with non-mutated GRIN2A were not available. Table S1 | Summary of NZM cell lines tested in this study, together with their mutation status and systemic therapy received by patients prior to cell line establishment.

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GRIN2A mutations in melanoma

Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Received: 02 November 2013; accepted: 30 December 2013; published online: 13 January 2014. Citation: D’mello SAN, Flanagan JU, Green TN, Leung EY, Askarian-Amiri ME, Joseph WR, McCrystal MR, Isaacs RJ, Shaw JHF, Furneaux CE, During MJ, Finlay GJ, Baguley BC and Kalev-Zylinska ML (2014) Evidence that GRIN2A mutations in melanoma correlate with decreased survival. Front. Oncol. 3:333. doi: 10.3389/fonc.2013.00333 This article was submitted to Cancer Genetics, a section of the journal Frontiers in Oncology. Copyright © 2014 D’mello, Flanagan, Green, Leung , Askarian-Amiri, Joseph, McCrystal, Isaacs, Shaw, Furneaux, During , Finlay, Baguley and Kalev-Zylinska. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Evidence That GRIN2A Mutations in Melanoma Correlate with Decreased Survival.

Previous whole-exome sequencing has demonstrated that melanoma tumors harbor mutations in the GRIN2A gene. GRIN2A encodes the regulatory GluN2A subuni...
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